Friday, December 6, 2013

Leucine, Insulin & Vitamin D*: A Hypertrophy Boosting Triplet That Does Not Make It From the Dish to the Gym? Evidence & Counter-Evidence from Human Trials

If you could simulate a workout at the beach in the petri dish, its beneficial health effect would be all the rage ;-)
I am pretty sure all of you still remember the recent post about the literal muscle building effect of vitamin D, right (see "Vitamin D Builds Muscle")? Me too, and so I was almost tricked to believe I was looking at the Girgis study, when I scanned the contents of the latest issue of Molecular Nutrition & Food Research, in which you'll with the telling title: "1,25(OH)2-vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes". Ah, ok, I see that's not exactly easy to understand. Well, in plain English this means as much as...

Calcitriol boosts the anabolic effect of leucine & insulin

If your read the "Vitamin D Builds Muscle" article, you should actually remember the dichotomous nature of the effects vitamin D had on muscle cell hypertrophy (which goes up) and proliferation (which goes down). This was after all the most intriguing result of the Girgis study (go back). The new data from the paper at hand, which is likewise dealing with in-vitro effects of vitamin D, does now provide us with some additional information on the underlying mechanisms of the hypertrophy effects.
Figure 1: Protein synthesis, insulin receptor expression and the levels of p-AKT, p-mTOR, and p-70S6K, all regulators of skeletal muscle protein synthesis in muscle cells with and without additional 1,25(OH)2D3 in a leucine + insulin filled Petri dish (Salles. 2013)
If we go by the data in Figure 1 it's obvious that the latter is driven by a direct amplifying effect of 1,25(OH)2D3, the active form of vitamin D, aka calcitriol, on the leucine and insulin induced increase in muscle protein synthesis. Unfortunately, this does not tell us a word about the potential consequences of the anti-proliferative effects of vitamin D on long-term increases in muscle size and the repair of damaged muscle fibers.
Vitamin D and health - What the latest systematic review says: As long as you look at things at the population level or sit in your well-climatized lab next to the Petri dishes, vitamin D is king. When you look at the real world, of which I still believe that it is populated by individuals, the excitement appears to be unwarranted. The most recent systematic review that has been published today in the (most) prestigious medical journal The Lancet says: "The discrepancy between observational and intervention studies suggests that low 25(OH)D is a marker of ill health. Inflammatory processes involved in disease occurrence and clinical course would reduce 25(OH)D, which would explain why low vitamin D status is reported in a wide range of disorders." (Autier. 2013) -- in other words: Being sick will lead to reduction ins 25(OH)D and not vice versa.
It is nevertheless intriguing to see, how the myotubes that had been cultured in 1,25(OH)2D3 solutions at 0, 1, or 10 nM for 72 h reacted to the leucine and insulin challenges. 14–16% increases in fractional protein synthesis rates (FSR) and an increased expression of insulin receptors, of which we can hope that it would also translate into increased glucose uptake into the cells. Assuming that similar effects occur in vivo and in a normal vs. high vitamin D environment, these benefits would be more than just statistically significant.

Ok, that's an in vitro study, so why do you discuss it at all?

If the above is what you've just been thinking, you know me quite well by now. I would indeed not have wasted a whole SuppVersity article on this vitamin D paper, if it would not contradict the real-world results of a recent randomized, double-blind, placebo-controlled study from the University of Oslo so "nicely".  In this paper, a group of Swedish researchers probed the effects of provision of 1,000IU and 400IU of vitamin D3 per day on 251 healthy adult men and women (age 18-50 years; Knutsen. 2013). 

Despite the fact that the vitamin D levels of the subjects almost doubled, none of the strength and performance parameters, i.e. jump height, handgrip strength and the chair-rising test, showed pre vs. post differences that differed from those in the control group.
Figure 2: Relative pre vs. post changes in jump height, grip strength and the time it took the subjects to complete the chair test; no statistically significant inter-group differences were observed (Knutsen. 2013) vs. relative increase in strength (no inter-group differences) in obese, vit D deficient resistance trained individuals (Carillo. 2013)
This in turn raises the question, whether all our new enthusiasm about 'vitamin D' (in the broad sense, i.e. D3, 25(OH)D3 and 1,25(OH)2D3) was misplaced. That's unquestionably a tricky question and if those "enthusiasm" was triggered by the idea that vitamin D would have independent anabolic or ergogenic effects, the answer is probably "Yes". If we are yet talking about the general role of vitamin D in the complex concert of muscular health, the answer must be "No!" There are, after all exceptions to the "vitamin D does not build muscle rule" that applies so nicely to the Knutsen study - there aren't many, but they are there.

The study Carillo et al., for example (Figure 2, right). If you take a look at the results , it's easy to see that for the twenty-three overweight and obese (age: 26.1±4.7 y; BMI: 31.3±3.2 kg/m², body fat: 43%) subjects with insufficient vitamin D levels (25-hydroxyvitamin D: 19.3±7.2 ng/ml) the researchers from the Purdue University recruited for their experiment the 4000IU of supplemental vitamin D each of them received on a daily basis had the expected effect on the resistance training induced increase in peak power and reduction in waist-to-hip ratio (not shown). The effect size is however is pathetic and the only thing that was "significant" - imho statistically, only - was the peak power in the vitamin D group.
A brief note on 25(OH)D vs. 1,25(OD)2D3: I know that it may sound as if it sucks that taking D3 supplements won't increase the amount of calcitriol in your veins, but if it did, I know more than a handful of people whose trust in some gurus would already have cost them a kidney or even their lives. There is a good reason that calcitriol is a prescription drug, because a dysregulation of the 1,25(OH)2D3 levels in the blood will increase the calcium deposition in the organs and vasculature (Bas. 2006) and would thus have the opposite effects most people expect from their high dose vitamin D3 supplementation regimen.
Bottom line: On paper, the real world-evidence from vitamin D deficient obese individuals in Carillo's recent study does supports the notion that vitamin D is an important facilitator of skeletal muscle protein synthesis, what it does not do, though is provide the missing conclusive evidence that being in the upper tertile of the physiological range (not restoring deficiencies as in Ceglia. 2013, for example) has beneficial real-world effects on muscle strength or size.

If you take another look at the doses the vitamin D enthusiasts from the Girgis study bathed their cells in, that the Swedes used active vitamin D, i.e. 1,25(OH)2D3 and that there is no direct relation between vitamin D3 intake, the serum levels of 25(OH)D3 and the amount of calcitriol (1,25(OH)2D3) your cells are exposed to, it's actually not surprising that the muscle building effects don't translate from Jérôme Salles' calcitriol saturated Petri dishes into the real world of the 251 participants of the Knutsen and the majority of the other vitamin D3 supplementation studies, is it?
  • Autier, P. et al. (2013) Vitamin D status and ill health: a systematic review. The Lancet Diabetes & Endocrinolog, Available online 6 Decembee
  • Bas, A., Lopez, I., Perez, J., Rodriguez, M., & Aguilera‐Tejero, E. (2006). Reversibility of Calcitriol‐Induced Medial Artery Calcification in Rats With Intact Renal Function. Journal of Bone and Mineral Research, 21(3), 484-490.
  • Ceglia, L., Niramitmahapanya, S., Morais, M. D. S., Rivas, D. A., Harris, S. S., Bischoff-Ferrari, H., ... & Dawson-Hughes, B. (2013). A randomized study on the effect of vitamin D3 supplementation on skeletal muscle morphology and vitamin D receptor concentration in older women. Journal of Clinical Endocrinology & Metabolism, jc-2013.
  • Knutsen, K. V., Madar, A. A., Lagerløv, P., Brekke, M., Raastad, T., Stene, L. C., & Meyer, H. E. (2013). Does Vitamin D Improve Muscle Strength in Adults? A Randomized, Double-blind, Placebo-controlled Trial Among Ethnic Minorities in Norway. Journal of Clinical Endocrinology & Metabolism, jc-2013.
  • Salles, J., Chanet, A., Giraudet, C., Patrac, V., Pierre, P., Jourdan, M., ... & Walrand, S. (2013). 1, 25 (OH) 2‐vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes. Molecular nutrition & food research.